Want EJ, O'Maille G, Smith CA, Brandon TR, Uritboonthai W, Qin C, Trauger SA, Siuzdak G.
Solvent-dependent metabolite distribution, clustering, and protein extraction for serum profiling with mass spectrometry. Anal ChemAnal ChemAnal Chem. 2006;78 :743-52.
AbstractThe aim of metabolite profiling is to monitor all metabolites within a biological sample for applications in basic biochemical research as well as pharmacokinetic studies and biomarker discovery. Here, novel data analysis software, XCMS, was used to monitor all metabolite features detected from an array of serum extraction methods, with application to metabolite profiling using electrospray liquid chromatography/mass spectrometry (ESI-LC/MS). The XCMS software enabled the comparison of methods with regard to reproducibility, the number and type of metabolite features detected, and the similarity of these features between different extraction methods. Extraction efficiency with regard to metabolite feature hydrophobicity was examined through the generation of unique feature density distribution plots, displaying feature distribution along chromatographic time. Hierarchical clustering was performed to highlight similarities in the metabolite features observed between the extraction methods. Protein extraction efficiency was determined using the Bradford assay, and the residual proteins were identified using nano-LC/MS/MS. Additionally, the identification of four of the most intensely ionized serum metabolites using FTMS and tandem mass spectrometry was reported. The extraction methods, ranging from organic solvents and acids to heat denaturation, varied widely in both protein removal efficiency and the number of mass spectral features detected. Methanol protein precipitation followed by centrifugation was found to be the most effective, straightforward, and reproducible approach, resulting in serum extracts containing over 2000 detected metabolite features and less than 2% residual protein. Interestingly, the combination of all approaches produced over 10,000 unique metabolite features, a number that is indicative of the complexity of the human metabolome and the potential of metabolomics in biomarker discovery.
Go EP, Wikoff WR, Shen Z, O'Maille G, Morita H, Conrads TP, Nordstrom A, Trauger SA, Uritboonthai W, Lucas DA, et al. Mass spectrometry reveals specific and global molecular transformations during viral infection. J Proteome ResJ Proteome ResJ Proteome Res. 2006;5 :2405-16.
AbstractMass spectrometry analysis was used to target three different aspects of the viral infection process: the expression kinetics of viral proteins, changes in the expression levels of cellular proteins, and the changes in cellular metabolites in response to viral infection. The combination of these methods represents a new, more comprehensive approach to the study of viral infection revealing the complexity of these events within the infected cell. The proteins associated with measles virus (MV) infection of human HeLa cells were measured using a label-free approach. On the other hand, the regulation of cellular and Flock House Virus (FHV) proteins in response to FHV infection of Drosophila cells was monitored using stable isotope labeling. Three complementary techniques were used to monitor changes in viral protein expression in the cell and host protein expression. A total of 1500 host proteins was identified and quantified, of which over 200 proteins were either up- or down-regulated in response to viral infection, such as the up-regulation of the Drosophila apoptotic croquemort protein, and the down-regulation of proteins that inhibited cell death. These analyses also demonstrated the up-regulation of viral proteins functioning in replication, inhibition of RNA interference, viral assembly, and RNA encapsidation. Over 1000 unique metabolites were also observed with significant changes in over 30, such as the down-regulated cellular phospholipids possibly reflecting the initial events in cell death and viral release. Overall, the cellular transformation that occurs upon viral infection is a process involving hundreds of proteins and metabolites, many of which are structurally and functionally uncharacterized.
Want EJ, O'Maille G, Smith CA, Brandon TR, Uritboonthai W, Qin C, Trauger SA, Siuzdak G.
Solvent-dependent metabolite distribution, clustering, and protein extraction for serum profiling with mass spectrometry. Anal Chem. 2006;78 :743-52.
Publisher's VersionAbstractThe aim of metabolite profiling is to monitor all metabolites within a biological sample for applications in basic biochemical research as well as pharmacokinetic studies and biomarker discovery. Here, novel data analysis software, XCMS, was used to monitor all metabolite features detected from an array of serum extraction methods, with application to metabolite profiling using electrospray liquid chromatography/mass spectrometry (ESI-LC/MS). The XCMS software enabled the comparison of methods with regard to reproducibility, the number and type of metabolite features detected, and the similarity of these features between different extraction methods. Extraction efficiency with regard to metabolite feature hydrophobicity was examined through the generation of unique feature density distribution plots, displaying feature distribution along chromatographic time. Hierarchical clustering was performed to highlight similarities in the metabolite features observed between the extraction methods. Protein extraction efficiency was determined using the Bradford assay, and the residual proteins were identified using nano-LC/MS/MS. Additionally, the identification of four of the most intensely ionized serum metabolites using FTMS and tandem mass spectrometry was reported. The extraction methods, ranging from organic solvents and acids to heat denaturation, varied widely in both protein removal efficiency and the number of mass spectral features detected. Methanol protein precipitation followed by centrifugation was found to be the most effective, straightforward, and reproducible approach, resulting in serum extracts containing over 2000 detected metabolite features and less than 2% residual protein. Interestingly, the combination of all approaches produced over 10,000 unique metabolite features, a number that is indicative of the complexity of the human metabolome and the potential of metabolomics in biomarker discovery.
Go EP, Wikoff WR, Shen Z, O'Maille G, Morita H, Conrads TP, Nordstrom A, Trauger SA, Uritboonthai W, Lucas DA, et al. Mass spectrometry reveals specific and global molecular transformations during viral infection. J Proteome Res. 2006;5 :2405-16.
Publisher's VersionAbstractMass spectrometry analysis was used to target three different aspects of the viral infection process: the expression kinetics of viral proteins, changes in the expression levels of cellular proteins, and the changes in cellular metabolites in response to viral infection. The combination of these methods represents a new, more comprehensive approach to the study of viral infection revealing the complexity of these events within the infected cell. The proteins associated with measles virus (MV) infection of human HeLa cells were measured using a label-free approach. On the other hand, the regulation of cellular and Flock House Virus (FHV) proteins in response to FHV infection of Drosophila cells was monitored using stable isotope labeling. Three complementary techniques were used to monitor changes in viral protein expression in the cell and host protein expression. A total of 1500 host proteins was identified and quantified, of which over 200 proteins were either up- or down-regulated in response to viral infection, such as the up-regulation of the Drosophila apoptotic croquemort protein, and the down-regulation of proteins that inhibited cell death. These analyses also demonstrated the up-regulation of viral proteins functioning in replication, inhibition of RNA interference, viral assembly, and RNA encapsidation. Over 1000 unique metabolites were also observed with significant changes in over 30, such as the down-regulated cellular phospholipids possibly reflecting the initial events in cell death and viral release. Overall, the cellular transformation that occurs upon viral infection is a process involving hundreds of proteins and metabolites, many of which are structurally and functionally uncharacterized.